Structural insights into the synthesis of FMN in prokaryotic organisms

Abstract

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyltransferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure of Corynebacterium ammoniagenes FADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-length CaFADS protomer and the quaternary organization.

Uptake and Metabolism of Antibiotics Roseoflavin and 8-Demethyl-8-Aminoriboflavin in Riboflavin-Auxotrophic Listeria monocytogenes

“…In contrast to Lmo0728 (RibC), B. subtilis RibR has a very low turnover number and does not contribute to flavin cofactor synthesis (38). Lmo0728 (RibC) misses two highly conserved domains present in all flavokinases (39), which explains why Lmo0728 (RibC) is not able to synthesize FMN from riboflavin and ATP (see Fig. S5 in the supplemental material).…”

The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes

“…Therefore, it was proposed that binding and dissociation of substrates/products of the Ca FADS enzymatic activities, FMNAT and particularly RFK, induce conformational changes which lead to self-association/dissociation processes. No dependence on the protein concentration for these processes has been observed in vitro 8, 9 .

Figure 1Structure of Ca FADS. ( A ) Cartoon representation of the Ca FADS monomer (PDB code: 2x0k) and of the dimer of trimers model predicted by the PISA server (one of the trimers is represented as a surface).

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“…Loop L4n (n denotes for N-terminal FMNAT module), loops L1c-FlapI and L6c, together with helix α1c, form the interface between two protomers within the trimer and contribute to the structures of the active sites (Figure 1A) 6 . L1c-FlapI, L6c and α1c, together with loop L4c-FlapII, form the closed conformation of the RFK flavin-binding site that is reached after being triggered by substrate/product binding (Figure 1B) 9 . In addition, a salt-bridge between the catalytic base at the RFK module, E268, and R66 at loop L4n stabilizes each trimer within the dimer of trimers 16, 19 , while residues at the RFK catalytic site modulate binding parameters and catalytic efficiency at the FMNAT active site 16 .…”

Structure, function, and mechanism of proline utilization A (PutA)

“…The metabolic logic of channeling in DMGO is likely the protection of the unstable iminium intermediate [56]. Also, prokaryotes have bifunctional FAD biosynthetic enzymes that use flavins as substrates in sequential riboflavin kinase and FAD synthetase reactions [57, 58]. …”

“…Substrate channeling occurs in many other enzymes [56, 57, 67–69], but whether the channeling step is hysteretic remains to be determined. Interestingly, a few channeling systems show possible indicators of hysteretic channeling (Table 4).…”